Synthesis of PAMAM Dendrimer Derivatives with Enhanced Buffering

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Synthesis of PAMAM Dendrimer Derivatives with Enhanced Buffering Capacity and Remarkable Gene Transfection Efficiency Gwang Sig Yu,† Yun Mi Bae,† Hye Choi,† Bokyung Kong,§ Insung S. Choi,*,§ and Joon Sig Choi*,†,‡ †

Department of Biochemistry Graduate School of Analytical Science and Technology Chungnam National University, Daejeon, 305-764, Korea § Molecular-Level Interface Research Center, Department of Chemistry, KAIST, Daejeon, 305-764, Korea ‡

bS Supporting Information ABSTRACT: In this study, we introduced histidine residues into L-arginine grafted PAMAM G4 dendrimers to enhance proton buffering capacity and evaluated the physicochemical characteristics and transfection efficacies in vitro. The results showed that the synthesized PAMAM G4 derivatives effectively delivered pDNA inside cells and the transfection level improved considerably as the number of histidine residues increased. Grafting histidine residues into the established polymer vector PAMAM G4-arginine improved their proton buffering capacity. The cytotoxicity of PAMAM G4 derivatives was tested and it was confirmed that they displayed relatively lower cytotoxicity compared to PEI25KD in various cell lines. Also, confocal microscopy results revealed that PAMAM G4 derivatives effectively delivered pDNA into cells, particularly into the nucleus. These PAMAM dendrimer derivatives conjugated with histidines and arginines may provide a promising polymeric gene carrier system.

’ INTRODUCTION The development of an effective and safe gene delivery carrier has become one of the most important factors enabling gene therapy. In the past decade, numerous studies have developed efficient transfection methods for delivering therapeutic genes into cells for human gene therapy. Viral and nonviral vector systems are the two major systems that transfer genetic material into cells. Currently, using adenoviruses and retroviruses to transfer genetic material (DNA or RNA) into cells is the general method in most ongoing gene therapy clinical trials. The viral vector system provides attractive advantages such as efficient transfection and high gene expression efficiency. However, there are some concerns such as immunogenicity, high toxicity, and the possibility of destructive integration into the human genome. Due to the disadvantages of viral vectors, the demands for alternatives to the viral vector system have increased.1,2 Although they display lower transfection efficiency than viral vectors, using nonviral vectors to deliver therapeutic genes into cells has several advantages. Nonviral vectors can deliver a relatively larger size of DNA into cells, and they have lower cytotoxicity and greatly reduced immunogenicity. Various methods are utilized to transfer genes into cells by nonviral approaches. Physical methods include a gene gun, electroporation, and hydrodynamic and ultrasound-mediated injection. These physical transfer techniques induce transient impacts on plasma r 2011 American Chemical Society

membranes, so that genetic materials can be introduced. Compared with physical approaches, using chemical methods to transfer genes has also been investigated. Cationic lipids condense pDNA, which is negatively charged due to its phosphate backbone. Many cationic lipid-mediated gene delivery strategies have been studied since a report published by Felgner et al. in 1987.3 Similar to cationic lipids, cationic polymers condense negatively charged pDNA through electrostatic interaction. Cationic polymers condense pDNA effectively and facilitate cellular uptake via endocytosis.4
8 Cationic polymers include polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, chitosan, dextran, protamine, and cationic polypeptides such as poly(L-lysine). PAMAM dendrimers are one of the most popular polymers used as a nonviral gene carrier because of their unique characteristics such as uniform size distribution, relatively higher transfection efficiency, and lower cytotoxicity compared to other traditional cationic polymers.9,10 Many studies have investigated the enhanced transfection efficiency of modified PAMAM dendrimer derivatives. Grafting of specific ligands for certain receptors (e.g., RGD sequence for Rvβ3 integrins) provides enhanced targeting effects to carriers.11 Moreover, the acetylation of PAMAM dendrimers decreases cytotoxicity Received: November 4, 2010 Revised: April 18, 2011 Published: May 02, 2011 1046

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Figure 1. Overall synthesis scheme of PAMAM G4-Hisn-Arg.

while maintaining membrane permeability.12 In our previous study, we synthesized an arginine-conjugated PAMAM dendrimer, which shows enhanced transfection efficiency compared to the native PAMAM dendrimer. 13 A PAMAM dendrimer has positively charged amine groups on the peripheral ends to allow interaction with the negatively charged pDNA phosphate backbone.14,15 Therefore, it is essential that newly designed vectors have cationic functional groups in their structure. Moreover, several physiological barriers must be overcome to achieve effective transfection.16 It is known that polyplexes could be internalized into cells by endocytosis forming endosomes.17 These endosomes progress into a lysosome state, which has an acidic environment to digest external materials. Therefore, most of the complexes in lysosomes will collapse. Thus, the proton buffering ability of the carrier is needed to overcome this acidic environment of the lysosome. Although PAMAM dendrimers have proton buffering abilities (hypothesized proton sponge effect) in their tertiary amine, insertion of additional functional groups that act as proton sponges is required to enhance endosomal release.18,19 Durability against the acidic environment should increase the number of complexes escaping from endosomes to the cytosol. The histidine conjugation method could be helpful for increasing buffer capacity against acidic conditions due to the presence of imidazole ring groups that have pKa of 6.0, which is a unique property that provides protonation ability in an acidic environment. Although native PAMAM dendrimers condense pDNA efficiently, additional conjugation with amino acids, such as histidine, arginine, and lysine, to the peripheral amine functional groups could contribute to their multifunctional properties. Particularly, histidine conjugation of a conventional polymer vector system has advantages not only in enhancing complexation ability but also in improving physicochemical strength against an acidic condition via the proton sponge effect. Some groups have reported applications of histidine as a proton buffering agent during liposomal delivery20,21 and polyhistidine as a proton sponge unit.22

In this study, we conjugated histidine and arginine residues to PAMAM dendrimers to obtain higher proton buffering capacity with ionizable imidazole groups on histidine units together with the high gene expression ability of PAMAM G4-Arg. We investigated the physicochemical properties, polyplex formation, cytotoxicity, and transfection efficiency of PAMAM G4 derivatives. The intracellular distribution of synthesized PAMAM G4 derivatives was also confirmed using confocal microscopy.

’ MATERIALS AND METHODS Materials. PAMAM generation 4, N,N-diisopropylethylamine (DIPEA), ammonium persulfate (APS), N,N,N0 ,N0 -tetramethylethane-1,2-diamine (TEMED), and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich (Seoul, South Korea). N-Hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1, 3,3-tetra-methyluronium (HBTU), and Fmoc-His(trt)-OH were purchased from Anaspec (San Jose, CA, USA), and Fmoc-Arg(pbf)-OH was obtained from Novabiochem (San Diego, CA, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and 100 antibiotic
antimycotic agent were purchased from Gibco (Gaithersburg, MD, USA). The luciferase expression plasmid was prepared as reported previously.23 The luciferase assay kit was purchased from Promega (Madison, WI, USA). The Micro BCA Protein Assay Kit was obtained from Pierce (Rockford, IL, USA). Synthesis of PAMAM G4-His(1,2,3)-Arg. Fourth-generation PAMAM was reacted with 4 equiv of Fmoc-His(trt)-OH, HBOt, and HBTU and 8 equiv of DIPEA in anhydrous DMF solution for 16 h at room temperature (R.T.). Then, the intermediate product was precipitated with an excess amount of cold ethyl ether. The precipitated intermediate was dissolved with 30% piperidine solution (v/v) and reacted for 1 h to deprotect the Fmoc group on the histidine unit. After the deprotection procedure, the intermediate product was precipitated with cold ethyl ether. Additional histidine elongation could be conducted by repeating the synthetic process described above. After elongation of the histidine unit, 4 equiv of 1047

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Bioconjugate Chemistry Fmoc-Arg(pbf)-OH, HOBt, and HBTU and 8 equiv of DIPEA were reacted with the intermediate product using the same method described above. After precipitation with cold ethyl ether, the intermediate product was reacted with 30% piperidine solution for 1 h to remove the Fmoc group on the arginine unit. Then, the product was washed and precipitated with cold ethyl ether. The precipitated product was dissolved with deprotection reagent (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/H2O, v/v, respectively) and stirred for 6 h at R.T to deprotect the pbf and trt groups on each amino acid unit. The unrefined product was dialyzed with distilled water using dialysis membrane (MWCO 3,500, Spectra/por) for 24 h to purify the product from unreacted amino acids or coupling reagents. After dialysis, the final product was freeze
dried. Overall schemes for the synthesis of PAMAM conjugates are presented in Figure 1. Agarose Gel Retardation Assay and Dynamic Light Scattering Analysis. A complex test was conducted to analyze the complex formation of PAMAM derivatives with plasmid DNA under various charge ratio conditions. Complexes were prepared containing polymers and plasmid DNA with different charge ratios by incubating them in HEPES buffer (25 mM, pH 7.4). A fixed volume of plasmid DNA and HEPES buffer with different polymer charge ratios was mixed at R.T. for 30 min. Each complex sample was analyzed on a 0.7% agarose gel containing ethidium bromide (0.5 μg/μL gel). Agarose gel electrophoresis typically required 30 min at 100 V. The size distribution and zeta potential of the polyplexes were measured using a Zetasizer Nano-Zs (Malvern Instruments, Worcestershire, U.K.). PAGE Study. Native polyacrylamide gel electrophoresis study of PAMAM derivatives was performed on a Mini-Protean Tetra Cell System (Biorad, Hercules, CA, USA). Citrate buffer (pH 3.0, 0.1 M) was used as the buffer system, and 6% T gels (T = g acrylamide þ g N,N0 - methylenebisacrylamide/100 mL solution) were prepared with a 30% acrylamide mixture solution (Biorad) with a combination of APS, TEMED, and Tris-HCl buffer (pH 7.5). The 6% T gels were acidified (pH 2) before adding the APS and TEMED using 5 N HCl to protonate the polymers and produce a narrow band. Reverse polarity (with the cathode at the bottom of the gel) was used for analysis of the positively charged PAMAM derivatives. Native PAGE typically required 50 min at 100 V. Polymer samples (1 μg) were prepared, and 1 μL of tracking dye (50% sucrose solution containing 1% methylene blue) was added to the samples for loading. Gels were stained overnight with 0.025% Coomassie Blue R-250 in 40% methanol and 7% acetic acid aqueous solution. After the staining process, the gels were destained with 10% (v/v) methanol and 10% acetic acid in water. Acid
Base Titration Assay. The protonation ability of PAMAM derivatives was determined by the acid
base titration method. PEI25KD (2 mg, 8  10
8 mol) and the same equivalents of PAMAM G4, PAMAM G4-Arg, and PAMAM G4-His(1,2,3)-Arg were titrated with 0.1 N HCl. NaCl (150 mM) and H2O were titrated as a control group. The pH value was determined with a pH-meter (pH211 microprocessor pH meter, HANA Instruments, Seoul, South Korea). Briefly, each titration sample was prepared by adding an equivalent amount of sample (8  10
8 mol) in 4 mL of 150 mM NaCl, and 100 μL of 1 N NaOH was added to the sample solution. Samples were titrated by adding aliquots (20 μL) of 0.1 N HCl until pH 3.0 was reached. Cytotoxicity Assay. The cytotoxicity of the PAMAM derivatives was assessed using the EZ-Cytox Reagent (Daeil Lab Service, Seoul, South Korea) based on the WST-1 method.

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Briefly, HepG2, 293, and NIH 3T3 cells were seeded in 96-well plates at a density of 2  104 cells/well except for HeLa cells (1  104 cells/well) and incubated in 100 μL of DMEM containing 10% FBS. After a 1-day incubation, the cells were treated with various concentrations of PEI25KD, PAMAM G4, and PAMAM derivatives. After further incubation for 24 h, 10 μL of EZ-Cytox Reagent was added to each well and incubated for an additional 4 h. Absorbance was measured at 450 nm using a VESAmax microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA). Confocal Microscopy Study. HeLa cells (5  103 cell/well) were seeded on culture slides and incubated at 37 °C. After a 24 h incubation, the cells were treated with polyplexes composed of various polymers with Alexa Fluor 532 labeled pDNA. Alexa Fluor 532 labeled pDNA was prepared according to the manufacturer’s protocol. After a further incubation for 24 h, old medium was removed, and the cells were washed with PBS. Cells were fixed in 4% paraformaldehyde for 10 min, and the nuclei were stained with 10 μg/mL bisbenzimide (Hoechst 33342) for 7 min and then washed with PBS several times. The plastic chamber on the glass slide was detached, and the cells were treated with mounting medium (Dako, Carpinteria, CA, USA) and covered with a coverslip. The fluorescent signal was analyzed using a Zeiss LSM 5 Live confocal laser microscope. Cell Culture and Transfection Assay. Human hepatocellular liver carcinoma HepG2 cells, human embryonic kidney 293 cells, murine fibroblast NIH3T3 cells, and human epithelial carcinoma HeLa cells were maintained at 37 °C in an incubator (5% CO2, 95% relative humidity). All cells were maintained in T25 flasks using DMEM supplemented with 10% FBS and 1% antibiotics. Cells were subcultured using a 0.25% trypsin/ethylenediaminetetraacetic acid solution when confluence reached 70
90%. For the transfection study, cells were seeded in a 24-well plate at a density of 2  105 cells/well except for HeLa cells (1  105 cells/ well) and incubated for 24 h to reach 70
80% confluency. The transfection complex was prepared by mixing 1 μg of plasmid DNA with various amounts of PAMAM derivatives containing 10% FBS for 30 min at R.T. To compare the transfection efficiency, PEI25KD/pDNA (polymer nitrogen(þ)/pDNA phosphate(
) (N/P) ratio: 7/1, optimal condition) and PAMAM G4/pDNA (N/P ratio: 4/1, optimal condition) complexes were prepared as a control group. PAMAM G4 derivatives were prepared with N/P ratios of 4:1 to 8:1 to compare gene transfection efficiency in terms of charge ratios. Then, cells were treated with the complex solution and incubated for 24 h at 37 °C. Old medium was removed, and the cells were washed with DPBS and lysed for 30 min using reporter lysis buffer (Promega). Luciferase activity was measured using Lumat LB 9507 (Berthold Technology, Bad Wildbad, Germany), and protein content was measured using the Micro BCA Protein Assay Kit (Pierce). Statistical Analysis. The statistical analysis was performed using the unpaired Student’s t-test (GraphPad Prism 5). Differences between groups were considered statistically significant at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

’ RESULTS AND DISCUSSION Synthesis of PAMAM G4-His(1,2,3)-Arg. This study was based on our previous report in which we demonstrated that the transfection efficiency of the PAMAM G4 dendrimer is enhanced by surface modification with L-arginine. It was confirmed that introducing L-arginine to the surface of the PAMAM dendrimer enhanced transfection efficiency.13 Some reports have indicated 1048

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Figure 2. Chemical structure of PAMAM G4-His3-Arg (quarter section). PAMAM G4 dendrimer core appears in black, histidines in red, and arginines in blue, respectively.

that arginine-rich peptides can deliver oligo DNAs and even proteins in vivo.24,25 Furthermore, many groups have reported that oligoarginine can serve as a membrane translocational signal.26 Meanwhile, in 2008, Asayama et al. presented carboxymethyl poly(L-histidine) as a pH-sensitive polypeptide to enhance the transfection efficiency of polyplex gene delivery.27 Recently, in 2009, Nakamura et al. reported that the conjugation of histidine with mannosylated cholesterol derivatives showed significantly higher cellular uptake in macrophages.28 More recently, in 2010, Song et al. introduced histidine residues into the TAT peptide and applied them to a magnetofection system, and the transgene expression via ternary magnetofection complexes displayed a 4-fold improvement in vitro.29 Many other groups have utilized polyhistidine to increase transfection efficiency.30 The histidine imidazole group has a pKa value of 6.0 and plays a role as a proton sponge in a slightly acidic environment. Furthermore, the escape of pDNA from the endosome is one of the important steps for successful nonviral transfection methods, and the proton buffering ability of the gene carrier is necessary to

increase transfection efficiency. Hence, in this study, we introduced histidine units into the L-arginine grafted PAMAM G4 dendrimer to enhance proton buffering capacity. The synthesized PAMAM G4-His(1,2,3)-Arg was confirmed by 1 H nuclear magnetic resonance (NMR) spectroscopy (Supporting Information, Figure S1). The number of histidine and arginine units conjugated to PAMAM G4 was calculated based on the 1H NMR data. Arginine units had characteristic peaks at 1.5 ppm and 3.76 ppm, whereas the histidine units showed characteristic peaks at 4.5 ppm, 6.8 ppm, and 7.6 ppm. From the 1 H NMR results, it was confirmed that more than 60 histidine and arginine units were conjugated to the surface primary amines of PAMAM G4. This number of conjugated units means that more than 95% of the surface amines were covered with histidine(1,2,3)-arginine peptide. The overall schematic synthesis process of PAMAM G4-His(1,2,3)-Arg is presented in Figure 1, and the structural representation of the histidine-arginine peptide conjugated to PAMAM G4 (a quarter section) is shown in Figure 2. 1049

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Figure 3. (A) Agarose gel electrophoresis retardation assay of pDNA by PAMAM G4 derivatives. pDNA only (lane 1); polymer/DNA charge ratio 1:1 (lanes 2, 5, and 8); 2:1 (lanes 3, 6, and 9); 4:1 (lanes 4, 7, and 10). Lanes 2
4, 5
7, and 8
10 represents PAMAM G4-His1-Arg, PAMAM G4-His2-Arg, and PAMAM G4-His3-Arg with pDNA, respectively. (B) Native PAGE analysis of PAMAM G4 derivatives. PEI25KD, PAMAM G4, PAMAM G5, PAMAM G4-Arg, PAMAM G4-His1-Arg, PAMAM G4-His2-Arg, and PAMAM G4-His3-Arg in lanes 1, 2, 3, 4, 5, 6, and 7, respectively.

Analysis of the Agarose Gel Retardation Assay. An agarose gel retardation assay was performed to confirm the complex formation of PAMAM derivatives with pDNA. Because the PAMAM derivatives have positively charged amine groups on their arginine units, they can interact with the negatively charged pDNA phosphate backbone. Each PAMAM derivative was complexed with a fixed amount of pDNA at an N/P ratio of 1:1 to 4:1. As shown in Figure 3A, PAMAM G4-His1-Arg and PAMAM G4-His2-Arg retarded pDNA at an N/P ratio of 1:1. However, PAMAM G4-His3-Arg showed complete retardation at an N/P ratio of 2:1. It is thought that the increased ratio of PAMAM G4-His3-Arg compared to His1 and His2 is due to an increase in molecular weight and a decrease in charge density. Molecular weight and charge density of polycationic polymers are the most significant factors for forming mature polyplexes. It is generally accepted that polycationic vehicles, which have a high charge density, condense pDNA more effectively.31 Furthermore, the distance between the DNA phosphate backbone and the polycation molecule is just as important to formation of polyplexes as the charge density.32 The pKa of the imidazole group on histidine is approximately 6.0; thus, these groups might remain poorly protonated in a physiological environment (pH 7.4). Hence, forming polyplexes with arginine residues on the peripheral end of a functional group occurs more often than that of histidine residues. It is thought that only a small portion of the protonated histidines participate in forming polyplexes, and that the surface arginines provide a main force for complexation with pDNA.

Figure 4. (A) Average diameter and (B) zeta potential values of pDNA and polymer/pDNA complexes. Data are expressed as mean ( standard deviation (n = 3).

Although grafting histidine units between the PAMAM G4 dendrimer and L-arginine may not play a major role in the formation of complexes, the results indicate that histidine-arginine peptide conjugated PAMAM derivatives could also effectively condense pDNA via electrostatic interactions at a relatively low N/P ratio. Size Measurement and Zeta Potential Analysis of Polyplexes. Delivering the naked plasmid DNA into cells is very limited due to enzymatic degradation, the polyanionic character of pDNA, and its large size. Cationic proteins, such as serum proteins, can bind with pDNA in the bloodstream, and enzymes such as DNase may degrade the pDNA. Therefore, safe and efficient introduction of DNA into cytoplasm and translocation into the nucleus are essential steps for successful gene therapy. In this study, the mean diameter and zeta potential values of each polyplex were examined at the optimum charge ratio. The charge ratio of PEI25KD was 7:1. PAMAM G4 and derivatives were examined at a charge ratio of 8:1. As shown in Figure 4A, the mean diameter of the naked pDNA was approximately 900 nm. The PEI25KD/pDNA complex showed a mean diameter of 201.4 ( 4.86 nm, and the native PAMAM G4 polyplex was 1050

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Bioconjugate Chemistry 169.53 ( 49.92 nm. Interestingly, the arginine-grafted PAMAM G4 dendrimer revealed a size of 102.64 ( 11.12 nm compared to native PAMAM G4. The sizes of the PAMAM G4-His1-Arg, PAMAM G4-His2-Arg, and PAMAM G4-His3-Arg polyplexes were 100.83 ( 22.81 nm, 111.83 ( 1.85 nm, and 125.9 ( 11.52 nm, respectively. From the results, the synthesized PAMAM derivatives, including PEI25KD and native PAMAM G4, formed nanometer-sized polyplexes effectively. As shown in Figure 4B, the zeta potential values of the PAMAM derivatives increased along with the elongation of histidine units. PEI25KD showed a highly positively charged zeta potential value of 30.6 ( 4.60 mV and that of PAMAM G4 was 21.73 ( 0.93 mV. As expected, arginine- and histidine-coupled PAMAM derivatives showed increased zeta potential values compared to native PAMAM G4. The zeta potential value for PAMAM G4-Arg was 24.93 ( 2.03 mV, and this increased value seemed to come from the increased surface charge of polyplexes. Similar results were revealed for PAMAM G4-His(1,2,3)-Arg with zeta potentials of 31.17 ( 3.46 mV, 33.73 ( 1.56 mV, and 37.47 ( 3.50 mV, respectively. As the guanidine group of arginine and the imidazole group of histidine have pKa of 12.0 and 6.0, respectively, the His(1,2,3)-Arg conjugated PAMAM derivatives displayed a positive charge under neutral conditions. Taken together, these results suggest that PAMAM derivatives can effectively condense pDNA into a nanometer size (around 100 nm) and possess zeta potential values of approximately 35 mV. The actual size and shape of each polyplex were further obtained using atomic force microscopy (AFM) (Supporting Information, Figure S2). PEI25KD showed a mostly globular shape, and some large polyplexes were also observed. Particularly, PAMAM G4-Arg had a very compact particle size (